Calcium homeostasis in the Central Nervous System – implications
for brain development and autism

Mechanisms related to calcium homeostasis that influence neuronal
growth, branching, differentiation, maturation, motiliy and
structural organisation in developing brain are reviewed in
this section, together with their possible role in the etiology
of autism.

Various neuropathological and MRI studies have pointed to the
following neurological abnormalities in autism:

* abnormalities of cortical development
has been observed in some cases, including areas of increased
cortical thickness, high neuronal density, neuronal disorganization
and poor differention of neurons.

Intracellular calcium homeostasis is essential for neuronal
development and function and calcium influx through voltgate
gated calcium channels (VGCC) regulates numerous processes in
the central nervous system (CNS), including neuronal growth,
differentiation, motility and excitability, secretion of neurotransmitters
and hormones, synaptic plasticity, neurotoxicity and neuronal
gene expression. Regulation of calcium entry through VGCC is
also of major importance in sensory processing and motor function.
Because of the critical role of calcium channels in signalling
processes, disruption of their function can lead to profound
disturbances in the structure and functioning of the nervous
system. Elevated levels of intracellular calcium are involved
in neurodegenerative mechanisms of the brain tissue and neurological
disorders can be caused by mutations in genes encoding calcium
channel subunits. There are currently several known human and
mouse channelopathies of the CNS, including a recessive retinal
disorder, X-linked congenital stationary night blindness, familial
hemiplegic migraine, episodic ataxia type 2, and spinocerebellar
ataxia. At present there are two known calcium channel genetic
mutations directly linked to autism (see Genetic-Factors).
Murine recessive neurological disorders as results of mutations
in genes encoding calcium channels include the tottering, leaner,
and rocker phenotypes with ataxia and absence epilepsy, and
the rolling Nagoya phenotype with ataxia without seizures.

Ion entry into neurons occurs either through receptor-operated
channels, for example GABA and NMDA channels, or through voltage-gated
ion channels. Although it is recognised that calcium entry through
both types of channels, as well as calcium relased from internal
stores [15709700]
may be important in the etiology of psychiatric disorders, this
review will mainly focus on the role of voltage gated calcium
channels, and L-type calcium channels (LTCC) in particular.

LTCC are localized on nerve terminals in the pre and postsynaptic
parts, as well as on cell bodies. Although many cells throughout
the body express LTCC, their densitity is higher in the brain,
especially during both development and aging. Apart from neurons,
calcium entry through LTCC is a major event in many processes
in both microglia and astrocytes, the supporting cells of the
CNS.

Calcium
homeostasis in developing brain

Neuronal cell development is controlled by a tightly organised
and regulated sequence of events that include cellular proliferation,
differentiation, migration and maturation. Signalling by calcium
ions plays a central role in these events.

Age-dependant changes in calcium signalling

VGCC are highly expressed during development and their function
is critical for developing neurons. In postnatally developing
brain, a transitional period for still developing neurons, there
appears to be a critical window in development in which disturbances
in calcium homeostasis may have significant consequences [16921238,
10493768].

In vitro neuronal cultures have exibited great differences in
sensitivity to changes in levels of intracellular calcium, depending
on the exact stage of development. These age-dependent changes
in functioning of VGCC have so far been linked to the onset of
several developmental events, including neuronal differentiation
[7515527],
neurite outgrowth and synaptogenesis [7790927,
7965045].
An age-dependent role of LTCC in developmental regulation of transmitter
phenotype in neurons has also been demonstrated, whereas the expression
of tyrosine hydroxylase (TH), a dopaminergic marker, in developing
neurons was shown to be dependent on the activities of LTCC [9437025]
(see Neurotransmitters).

Neuronal gene expression

A transcription factor is a protein that acts as a regulator of
gene expression. CREB (cAMP response element-binding) proteins
are transcription factors which bind to cAMP response elements
in DNA and thereby increase or decrease the transcription of certain
genes. CREB has been widely studied due to its role in diverse
functions such as circadian rhythms, drug addiction and inflammatory
pathways. Both CREB and several transcriptional regulators have
been linked to epigenetic factors involved in cognitive and behavioural
developmental disorders [15721740].
CREB deficient mice for example were shown to exibit less active
and exploratory behaviours in novel environments, as well as memory
deficits in spatial learning and fear conditioning [15233759,
15805310].

One of the ways in which calcium channels influence neuronal and
many other activities is via signaling pathways that control gene
expression. This involves regulation of various transcription
factors, including CREB. Calcium entry specifically through LTCC
is particularly important for transcriptional responses in neurons,
muscle, pancreatic beta cells and osteoblasts. Through
its stimulation of CREB nuclear calcium may modulate the expression
of numerous genes including neurotransmitter receptors and transmembrane
and scaffolding proteins, with the involvement of most having
been implicated in autism (see Neurotransmitters
and Genetic-Factors). LTCC
in brain have been implicated in mediating many long term changes
in neuronal activity, some having behavioural and cognitive modulation
as their end results. This importance of LTCC function in gene
expression has been observed in many diverse neurons, including
those found in the hippocampus, cortex, striatum, retina, dorsal
root ganglia and cerebellum. Early developing Purkinje neurons
prior to the stage of dendritic development express a somatic
calcium signaling pathway that communicates information from the
cell membrane to the cytosol and nucleus [16035195]
(see next).

Amongst other effects, opening of LTCC leads to CREB induced expression
of brain derived neurotrophic factor (BDNF)
and neuronal nitric oxide synthase (nNOS)
[11572963,
14604759].
nNOS activity regulates the production of nitric oxide, excessive
levels of which can be damaging to neurons, causing oxidative
stress and cell death. Calcium channel mutant mice that display
similarities with human neurological conditions, including autism,
all exhibit varying degrees of cerebellar dysfunction and neuronal
cell death, thought to be at least partly due to abnormal nNOS
[12834873].

With regards to BDNF, its levels and levels of BDNF autoantibodies
are known to be elevated in brains of individuals with autism,
with one study observing them to be three times higher than controls,
with on the other hand significantly reduced blood levels in adults
with autism [16181614,
11431227,
16876305].
Excessive activation of LTCC causes granule cells to express BDNF,
the release of which stimulates tyrosine kinase receptors (Trk)B
to induce axonal branching, which may establish hyperexcitable
dentate circuits implicated in epilepsy [15317847].
Exploring TrkB partial agonists as a possible treatment option
for autism has been suggested [16023301].
The mechanism of calcium and BDNF signalling also plays a role
in establishing granule cell synaptic transmission, including
levels of expression of NMDA receptors, during cerebellar development
[16221864].

Apart from BDNF, its neurotrophin family includes the growth factors
Nerve Growth Factor (NGF) neurotrophin 3 (NT-3), and neurotrophin
4 (NT-4), some of which were also found to be elevated in autism
in several studies [11357950,
16289943].
The same studies observed raised levels of neuropeptide vasoactive
intestinal peptide (VIP) compared to controls. The expression
level of VIP is influenced by calcium influx through LTCC, possibly
through similar mechanisms [15197736].
On the other hand VIP is able to influence VGCC conductance through
its known interaction with G-protein-coupled receptors [8772132,
15109935].

In addition, significant elevations of neuropeptide vasopressin
(AVP), with concurent reductions in levels of apenin,
a neuropetide that could counteract AVP action, have been observed
in autism. Again the involvement of raised calcium levels and
CREB activities has been suggested in the expression of vasopressin
gene [3607454,
9389510].

Possible involvement of Homer and Shank
protein complexes in the LTCC activation of CREB has been suggested
[15689539,
12716953],
as localized calcium responses, regulated by interactions with
PDZ domain proteins, are deemed necessary for this activation.
It should be mentioned that loss of the SHANK3/PROSAP2 gene has
been proposed to be responsible for the main neurological developmental
deficits observed in 22q13 deletion syndrome, characterised by
delays in speech and motor deveopment [16284256]
(see Genetic-Factors). Chromosomal
deletions of SHANK3 have recently been identified in a small number
of individuals with autism.

With reference to CREB-related activities possibly being relevant
in the etiology of autism, it should be added that sex hormone
estradiol has been noted to regulate CREB activity
via its direct and/or indirect effect on LTCC, and that considerable
overlap between behaviors and processes reliant on CREB and those
that are influenced by estradiol has been noted [15901789]
(see Gender Differences).

A reduced MET gene expression has been implicated
in autism susceptibility. A study analysis of the gene encoding
the pleiotropic MET receptor tyrosine kinase observed a decrease
in the promoter activity of the gene and altered binding of specific
transcription factor complexes in autism sample [17053076].
MET signaling plays a role in neuronal growth and maturation as
well as in the immune function and gastrointestinal repair, two
areas with frequently reported medical complications in autism.
The expression of tyrosine kinase receptors is linked to calcium
signalling pathways, and sudden changes in the levels of intercellular
calcium from both external and internal sources results in changes
in levels of MET tyrosine phosphorylation. This regulatory effect
of calcium is mediated through calcium-linked proteins and enzymes
[1651934,
2111905,
2005882].

It has been suggested that an important subset of developing hippocampal
interneurons expressing inhibitory GABA and GAD enzymes
express LTCC and that these channels likely regulate the development
of these interneurons [16154277,
11085875],
as well as the expression levels of GAD and GABA (see Neurotransmitters).

Increasing evidence suggests that the observed down-regulation
of Reelin mRNA in neurological disorders may
be caused by the dysfunction of epigenetic regulatory pathways
in these interneurons [17065238].
Reelin is a protein that is found mainly in the brain and that
acts on migrating neuronal precursors and controls correct cell
positioning in several areas of the brain. It is secreted by Cajal-Retzius
cells and by the external granule cell layer in the cerebellum
and its release rate depends solely on its synthesis rate. Abnormalities
in the expression levels of Reelin protein and mRNA, as well as
those ofr Reln receptor VLDLR in frontal and cerebellar areas
of autistic brains versus control subjects have been observed,
implicating impaired Reelin signalling in autism [15820235].
Several linkage studies have so far failed to establish a firm
genetic basis of this abnormality [15048647,
15048648].

It merits a mention in this context that the level of Reelin can
be affected significantly following exposure to x-radiation [10744063].
Whether the effect of x-radiation on calcium channel conductance
could be one likely mechanism behind this effect remains to be
established [9096258].
Of equal interest is the observation that in rodents prenatal
viral infection leads to significant reduction in production of
Reelin [10208446]
(see Viruses).

In addition to CREB, LTCC activate a number of other transcription
factors such as NFAT, MEF-2, and SRF. The nuclear factor
of activated T-cells (NFATc) was originally characterized in the
immune system, but is now known to play an important role in brain
function as well [link].

Another suggested mechanism for this privileged role of calcium
channels in epigenetic pathways is the role of the calcium
channel-associated transcription regulator (CCAT). CCAT
binds to a nuclear protein and in this way regulates the expression
of a wide variety of genes involved in neuronal signaling and
excitability. The nuclear localization of CCAT is regulated both
developmentally and by changes in intracellular calcium. If confirmed,
this mechanism would provide a more direct way in which VGCC activate
gene transcription in excitable cells [17081980].

Purkinje neurons

Purkinje cells are a class of GABAergic neurons located in the
cerebellar cortex. These cells exhibit a highly intricate dendritic
arbour, with a large number of dendritic spines. These cells are
of central importance for bodily functions of balance and coordination.
Cerebellar abiotrophy is a condition affecting some animals in
which Purkinje cells begin to atrophy shortly after birth, and
this often results in symptoms such as ataxia, intention tremors,
hyperreactivity, stiff or high-stepping gait, apparent lack of
awareness of where the feet are, and a general inability to determine
space and distance. A similar condition known as cerebellar hypoplasia
occurs when Purkinje cells either fail to develop or die prenatally.

The proliferation and survival of GABAeric neurons, including
Purkinje neurons, in developing brain seems to be dependent on
highly regulated calcium influx through VGCC. Increasing or decreasing
calcium currents through these channels was observed to have profound
effects on survival of cell cultures. This rate of dependence
and survival seems to be linked to the exact stage of development
(see above) [10366697].
As an illustration, the age-dependent effect of ethanol, a toxic
environmental factor, on developing Purkinje neurons is well known,
with ethanol being able induce to mitochondrial damage and ultimately
cell death [12204202].
These effects are likely due to ethanol-induced changes in VGCC
function and altered calcium signalling [16555300](see
Mitochondria).

Young and undeveloped Purkinje neurons without dendrites in culture
express only the high-threshold calcium current, which increases
approximately by half in amplitude during development, thus indicating
the importance of calcium conductances in development and maturation
of early Purkinje neurons [1377238].
One prominent function of calcium entry through LTCC in these
neurons is that this signalling pathway appears to convey developmental
cues directly to the nucleus, thus influencing activation of gene
transcription factors, CREB in particular (see above), and expression
of cellular proteins. This effect can be additionally amplifed
by release of calcium from intercellular stores [16035195,
16555300,
11007898].

Calcium signaling regulates both axonal and dendritic branching
in most types of developing neurons, including Purkinje neurons
[11248350].
Some of the mechanism of this effect is via abovementioned calcium-dependent
activation of CREB, its effects on cytoskeleton and its regulation
of the expression levels of neurotransmitters and neurothrophins
and activation of protein tyrosine kinases [15581694,
15882639,
8845164].
Calcium regulation of neurite growth and growth cone motility
is a process that is dependent on activation G-proteins and is
sensitive to pertussis toxin treatment.

Optimum levels of calcium influx promote normal dendritic and
axonal elongation and growth cone movements, which are involved
in neuronal directional pathfinding and target recognition. These
activities are essential for assembly of functional circuits within
the developing nervous system and for regeneration following damage.
Changes in levels of intercellular calcium and its way of entry
into the cells thus can have profound effects on the structure
and function of these neuronal networks [3121806].
Calcium transients regulate growth cone advance by direct effects
on the growth cone. These transients are mediated primarily by
LTCC and silencing them with channel blockers can in some circumstances
promote axon outgrowth [12574421].
Several factors that are though to influence neurite outgrowth,
for example serotonin and acetylcholinesterase,
are suggested to exert that influence through activities of LTCC
[12031351, 2376732,
10437116]. Brain
serotonin levels play an important role in developing brain and
impairments of serotonin metabolism have been implicated in autism.
In addition, a recent study has described maternal serotonin levels
as being of central importance for developing fetal brain (see
Maternal_Factors). In addition, significant preturbations
in brain levels of tryptophan and/or serotonin and its receptors
have been recorded in some viral infections [8158981,
3509812]
(see also Viruses). Several
in vitro studies have noted the inhibitory effects of serotonin
and its receptors on the function of VGCC and neuronal migration
during development [11976386,
11494406,
12401168].
On the other hand, calcium signals and functioning of VGCC play
an important role in serotonin metabolism and secretion, and in
the regulation of serotonin receptors expression and function
(see Neurotransmitters).

It has been suggested that calcium signals through LTCC influence
differentiation of neural stem/progenitor cells (NSC).
A study looking at NSC derived from the brain cortex of postnatal
mice observed that their differentiation is strongly correlated
with the expression of LTCC, and that influx of calcium ions through
these channels plays a key role in promoting neuronal differentiation
[1094458,
16519658].

In addition, calcium transients also play a central role in controlling
migration and organisation of both neuronal and
non-neuronal cells in the developing CNS [15820385,
16720042,
16029198,
15712206].
In vitro, neurons migrate in association with nonneuronal cells
to form cellular aggregates. Changes in those cell complexes in
cultured embryonic chick ciliary ganglion were observed in response
to treatments that increased or decreased intracellular calcium
concentration. Application of thimerosal, a compound that stimulates
calcium mobilization from internal stores, increased the amplitude
of spontaneous nonneuronal oscillations and the area of migrating
nonneuronal cells as well as the velocity of the neuronal-nonneuronal
cell complex [16720042].

In mouse ‘weaver’ phenotype the genetic mutation impairs
migration of the cerebellar granular neurons and induces neuronal
death during the first two weeks of postnatal life. Upregulation
of calcium channels was found to contribute to the migration deficiency
of these neurons. Loss of these neurons could be attenuated by
application of LTCC blockers [8707831].

Increases in intracellular calcium levels via upregulation of
VGCC activates calcium-calmodulin-dependent protein kinase (CaMK)
and calcineurin phophatase (CaN), which play an important role
in development and synaptic organization of granule cells during
early postnatal period [16793900].

Neuronal apoptosis

LTCC involvement in neuronal apoptosis (cell death) is probably
at least partly due to mitochondrial injury induced by excessive
calcium influx and ROS (see Mitochondria
and Oxidative Stress). Several
LTCC antagonists are able to attenuate cell injury and death in
culture, as being induced either by some pathogens, including
Amyloid beta protein implicated in etiology of Alzheimer’s
disease [15303126,
16321794,
15006551,
10964602]
(see Related Disorders).

Synapse formation and synaptic plasticity

It has been hypothesised that autism and related symptoms could
in part be a result of disruption of synaptic plasticity in developing
brain [15362161].
Synaptic plasticity is the ability of the synapses between two
neurons to change in strength. One of the mechanism underlying
synaptic plasticity involves regulation of gene transcription
and changes in the levels of key proteins at synapses [9437025].
Several recent studies have established a role of LTCC in long-term
potentiation (LTP), a long lasting enhancement in efficacy of
the synapses between the neurons, thought to be the cellular basis
of learning and memory [16251435].
One good example is cpg15, a gene that encodes a membrane-bound
ligand that regulates neurite growth and synaptic maturation,
and whose expression level is thought to be at least partly influnced
through LTCC activation of CREB [14664806].
LTCC are crucially involved in regulation of synapses of auditory
inner hair cells, which includes regulation of the expression
of potassium channels on those synapses [16828974]
(see Motor/Sensory).

Calcium homoestasis in neuroglia

Glial cells are cells that provide support and nutrition to neurons.
Astrocytes, the largest and most abundantly expressed
glial cells, form connective tissue of the brain and carry out
various functions, including induction of neuronal growth and
differentiation, participation in maintenance of blood brain barrier
and cerebral blood flow, regulation of ion concentration in the
extracellular space and modulation of synaptic transmission. Astrocytes
accummulate in areas where neurons have been damaged (gliosis).
Microglia are macrophages that have immunoprotective role in the
brain and play an important role in inflammatory responses.

Changes in intracellular calcium levels are an important signal
for communication between glial cells and neurons, and recent
evidence points to voltage gated calcium channels as playing an
important regulatory role in these processes. For example transient
increases in calcium levels in astrocytes either from external
sources or from internal stores can result in release of glutamate
and modulatation of synaptic transmission in surrounding neurons
[14966867, 12555202].
Furthermore, reactive gliosis as well as glial cell injury and
death is thought to be mediated by upregulation of VGCC [9502793,
9736645].
Astroglial release of proteins which enhance neuronal survival
and induce neuronal growth and differentiation can be blocked
by calcium antagonists and mimicked by Bay K 8644, a calcium channel
agonist, indicating the importance of calcium homoestasis in these
events [1397177].

For importance of calcium homeostasis in regulation of cerebral
blood flow as well as maintenance of blood brain barrier by astrocytes
see BBB.

Microglia play an important role in CNS inflammatory
responses, and its migratory and secretory responses can be modulated
by increases of calcium via LTCC. Several proteins and lipopolysaccharides
are know to be able to exert such influence, either directly or
through activation of chemokine receptors (see Immune/Inflammation)
[10858625,
9914452,
12805281].
Similar mechanism of rises in calcium levels following activation
of chemokine receptors has been observed in oligodendrocytes,
whose main function is to myelinate axons [16095689].
Stimulation of CXCR4 receptors and subsequent elevation of calcium
is in these neuroglial cells is a G protein-linked event [16837851].
It may be of interest in this context that an experimental mouse
model of multiple sclerosis was succesfully treated with calcium
antagonists bepridil and nitrendipine [15296830].
Furthermore, It has been proposed that oligodendrocytes, alongside
astrocytes, play an important role in regulating potassium levels
(see Epilepsy/Seizures).